The central nervous system (CNS) is composed of diverse neural cell types. A common paradigm in the development of the CNS is that single pools of neural progenitor cells in the neural epithelium give rise to the diverse neural cell types. Despite significant progress, a clear understanding of the genetic mechanism underlying formation of the cellular diversity in the CNS is still lacking. We use the mouse neural retina to address this question. Our long-term goal is to understand how transcription factors regulate gene expression globally to orchestrate the formation of the various retinal cell types. This proposal focuses on the fate- specification and differentiation of retinal ganglion cells (RGCs). As with other retinal cell types, RGC development initiates from natve multipotent retinal progenitor cells (RPCs), progresses in a stepwise fashion, and is regulated by a hierarchical gene regulatory network (GRN). Within this GRN, three transcription factors, Math5, Isl1 and Pou4f2, occupy key node positions at two different stages of RGC development. Math5 is upstream and is required for RPCs to gain competence for an RGC fate, whereas Isl1 and Pou4f2 are downstream and function collaboratively to initiate and maintain the gene expression program in RGC differentiation. However, the genetic and molecular basis for the specification of the RGC fate, a key aspect of RGC development, remains unknown. This grant proposal focuses on the roles of Isl1 and Pou4f2 in this process. Our central hypothesis is that Isl1 and Pou4f2 interact with each other and participate in RGC fate-specification and differentiation. This hypothesis is based on our current knowledge of defective development of RGCs, and corresponding alterations in gene expression, in the respective knockout mice. The experiments we propose will directly test this hypothesis from several different aspects. The specific aims we propose include: 1) To examine whether Isl1 and/or Pou4f2 can specify RGC fate in the absence of Math5; 2) To investigate the molecular basis for the Isl1/Pou4f2 collaboration in regulating downstream genes; 3) To map the binding sites of Isl1 and Pou4f2 in the genome of developing RGCs by ChIP-seq. Collectively, these experiments will provide significant new insights into the genetic pathways regulating RGC formation. By learning how RGCs form and are maintained, we will also obtain valuable information on why they die under certain disease conditions, such as glaucoma, optic neuritis, and ischemic optic neuropathy, and thus be able to develop novel preventive and therapeutic strategies for these diseases.